In this practical you will perform network modelling using fMRI timeseries data (i.e. the output from stage 1 of dual regression). The toolbox you will use for this is FSLNets, which currently requires MATLAB (or Octave - a free alternative).
To estimate networks and perform a statistical analysis on this requires the following steps:
In the next sections we will go through an example of how to run these steps. For this practical we re-ran for you the group ICA and the dual regression using 100 components, with the following command (please do NOT run this again!):
melodic -i input_files.txt -o groupICA100 \ --tr=0.72 --nobet -a concat \ --bgimage=$FSLDIR/data/standard/MNI152_T1_2mm_brain.nii.gz \ -m $FSLDIR/data/standard/MNI152_T1_2mm_brain_mask.nii.gz \ --report --Oall -d 100
The main thing you will feed into FSLNets network modelling is the timecourses obtained from stage 1 of a dual regression. Open a terminal and cd to the working directory of this practical, and then start octave:
cd ~/fsl_course_data/rest/Nets octave
Add the folder containing the scripts we are going to use to the path, and setup a few variables:
addpath ~/fsl_course_data/rest/FSLNets addpath(sprintf('%s/etc/matlab',getenv('FSLDIR'))) addpath ~/fsl_course_data/rest/octave/statistics-1.2.4/inst addpath ~/fsl_course_data/rest/octave/libsvm/matlab
Now we are ready to start setting up some important parts for our network modeling analysis. To load in all subjects' timeseries data files from the dual-regression output directory, run:
ts = nets_load('groupICA100.dr', 0.72, 0);
We will now have a quick look at the temporal spectra of the RSNs, as a quick check that these look reasonable. Running the following command will show you a figure in which the left part of the plot shows one spectrum per group-ICA component, each averaged across all subjects. The right part shows the same thing, but with all spectra overlapping.
ts_spectra = nets_spectra(ts);
There is now the option to remove components' (nodes') timeseries that correspond to artefacts rather than plausible nodes. Similar to the last practical, you need to decide this by looking at the spatial maps, timecourses, and frequency spectra. To save time, we have listed the good components for you. Run the following commands to list the good components and apply the cleanup:
ts.DD = [1:3,5,6:9,11:13,17:23,25:38,40,42,43,47:50,52,53,55:59,61,... 62,64:66,70:74,77,80,81,86,87,93,97]; ts = nets_tsclean(ts,1);
Now you are ready to compute a network matrix for each subject, which is in general a matrix of connection strengths (of size number of nodes by number of nodes). We will compute two different versions of these netmats for each subject: a simple full correlation ('corr'), and a partial correlation that has been regularised in order to potentially improve the mathematical robustness of the estimation ('ridgep'). The partial correlation matrix should do a better job of only estimating the direct network connections than the full correlation does.
Fnetmats = nets_netmats(ts,1,'corr'); Pnetmats = nets_netmats(ts,1,'ridgep',0.1);
The full and partial netmats are now calculated for all subjects. In the next section, you are going to compute the group average and take a look at the average network matrix.
Now that we have computed the full and partial network matrices for each individual subject. The next step is to perform a simple group-level analysis to look at the mean network matrix across all subjects. This can be done using the command below, which saves out both the simple average (Mnet) and the results of a simple one-group t-test across subjects as Z values (Znet).
The second input to this command decides whether or not to display a summery figure. This is set to 1 for the second command run on the partial netmats, so the summary figure for this will show up. On the left, this figure shows the results from the group t-test, and on the right is a consistency scatter plot showing how similar the results from each subject are to the group (i.e. the more this looks like a diagonal line, the more consistent the relevant netmat is across subjects).
Mnet_Pin row 3, column 27 is 6.59 (you can check this by typing
Mnet_P(3,27)into Octave and hitting enter). What does this number represent?
The next thing we can look at is how nodes cluster together to form larger resting state networks. For this we run a clustering method that groups nodes together based on their timeseries. To view this network hierarchy, run:
You can see, for example, that the nodes group together in the dark blue tree on the far left are part of a large-scale resting state network called the default mode network that you may have heard about.
We are now able to test whether the netmats differ significantly between
healthy controls and patients with a tumor using a two-sample t-test. This is
a 'univariate' test, as we will test each network matrix edge separately for a
group-difference, and then we will estimate p- values for these tests,
correcting for the multiple comparisons across all edges. By analogy to
high-level task-fMRI analyses: you can think of each subject's netmat as being
an NxN image of voxels, and the univariate testing as modelling each voxel (in
isolation from each other) across subjects. To perform the comparison please
run (which calls
randomise from within Octave, with 5000
permutations for each contrast):
Once randomise has finished, you will see a figure showing "netmats" containing corrected p-values. The results above the diagonal show edges where the two-group t-test is significant, at corrected-p<0.05.
We will now run a command that shows which nodes were linked to the significant result:
In addition, we also want to show how the partial correlation differs between the patients and the controls and these two significant edges. To do this, run:
The boxplots summarize the distributions of the correlation values (connections strengths) in the two groups - A being healthy controls and B being tumour patients - for this one particular node-pair (57,33).
exit in the terminal to exit octave if you are finishing the practical here and not doing the optional section below.
Finally, we will now carry out multivariate cross-subject analysis - meaning that, instead of considering each netmat edge in isolation (like we did above) we will consider the whole netmat in one analysis.
For example, we can attempt to classify subjects into patients or controls using machine learning methods, such as support vector machines (SVM) or linear discriminant analysis (LDA). Such methods look at the overall pattern of values in the netmat, and try to learn how the overall pattern changes between the two groups.
The following command feeds the regularised partial correlation netmats from both groups into LDA. It uses a method known as leave-one-out cross-validation to train and test a classifier, and reports in what percentage of tests it was successful at discriminating between patients and controls:
You can also try using other classifiers, for example by changing the last entry from 2 to 1 (type help nets_lda for further details).
One "downside" of such multivariate testing is that you can no longer make strong statistical claims about individual edges in the network - the whole pattern of edges has been used, so we don't know which individual edges are significantly different in the two groups.
exit in the terminal to exit octave.